Profile of Human Milk Phospholipids at Different Lactation Stages with UPLC/Q-TOF-MS: Characterization, Distribution, and Differences.

Human milk phospholipids are important for the regular growth and development of infants. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was employed to qualitatively and quantitatively analyze 277 phospholipid molecular species in 112 human milk samples to obtain a detailed profile of human milk phospholipids along the lactation stage. MS/MS fragmentation patterns of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were characterized in detail. Phosphatidylcholine is the most dominant group, followed by sphingomyelin. PC(18:0/18:2), SM(d18:1/24:1), PE(18:0/18:0), PS(18:0/20:4), and PI(18:0/18:2) showed the highest average concentration among all of the phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol molecular species, respectively. The fatty acids attached to the phospholipid molecules were mainly palmitic, stearic, oleic, and linoleic acids, and the plasmalogens decreased along the lactation stage. The increase of sphingomyelins and phosphatidylethanolamines and the decrease of phosphatidylcholines are the key changes from colostrum to transitional milk; the increase of lysophosphatidylcholines and lysophosphatidylethanolamines and the continuous decrease of phosphatidylcholines are the vital changes from transitional milk to mature milk.

Characterization of D-amino acids in colostral, transitional, and mature preterm human milk.

D-Amino acids are regulatory molecules that affect biological processes. Therefore, being able to accurately detect and quantify these compounds is important for understanding their impact on nutrition and health. There is a paucity of information regarding D-amino acids in human milk. We developed a fast method for simultaneous analysis of amino acid enantiomers in human milk using liquid chromatography with tandem mass spectrometry. The method enables the separation of 41 amino acids without chemical derivatization. Our results revealed that human milk from mothers of preterm infants contains concentrations of D-amino acids that range from 0.5 to 45% that of their L-counterparts and that levels of most D-amino acids decrease as the milk production matures. Moreover, we found that Holder pasteurization of milk does not cause racemization of L-amino acids. To our knowledge, this is the first study to describe percentages of D-amino acid levels in human milk; changes in D-amino acid concentration as the milk matures; and the effect of Holder pasteurization on D- and L-amino acid concentrations in human milk.

Vitamin A Concentration in Human Milk: A Meta-Analysis.

Humans require vitamin A (VA). However, pooled VA data in human milk is uncommon internationally and offers little support for dietary reference intake (DRIs) revision of infants under 6 months. As a result, we conducted a literature review and a meta-analysis to study VA concentration in breast milk throughout lactation across seven databases by August 2021. Observational or intervention studies involving nursing mothers between the ages of 18 and 45, with no recognized health concerns and who had full-term infants under 48 months were included. Studies in which retinol concentration was expressed as a mass concentration on a volume basis and determined using high-, ultra-, or ultra-fast performance liquid chromatography (HPLC, UPLC, or UFLC) were chosen. Finally, 76 papers involving 9171 samples published between 1985 and 2021 qualified for quantitative synthesis. Results from the random-effects model showed that the VA concentration of healthy term human milk decreased significantly as lactation progressed. VA (µg/L) with 95% CI at the colostrum, transitional, early mature and late mature stages being 920.7 (744.5, 1095.8), 523.7 (313.7, 733.6), 402.4 (342.5, 462.3) and 254.7 (223.7, 285.7), respectively (X2 = 71.36, p < 0.01). Subgroup analysis revealed no significant differences identified in VA concentration (µg/L) between Chinese and non-Chinese samples at each stage, being 1039.1 vs. 895.8 (p = 0.64), 505.7 vs. 542.2(p = 0.88), 408.4 vs. 401.2 (p = 0.92), 240.0 vs. 259.3 (p = 0.41). The findings have significant implications for the revision of DRIs for infants under six months.

Total and Sn-2 Fatty Acid Profile in Human Colostrum and Mature Breast Milk of Women Living in Inland and Coastal Areas of China.

INTRODUCTION: Although lipid is the major energy source and exerts beneficial effects on infant growth, research on the composition of fatty acid (FA) at the sn-2 position of human milk (HM) in China and abroad is limited. OBJECTIVES: This study aimed to investigate the FA positional distribution in colostrum and mature HM of women living in the inland and coastal areas of China and explore the potential influences of geographical region and lactation stage on the FA profile of Chinese women. METHODS: Colostrum milk (n = 61) and mature milk (n = 56) samples were obtained longitudinally from healthy lactating women in Guangzhou and Chengdu, China. Gas chromatography was used to determine the total and sn-2 FA composition. RESULTS: Significant differences were observed in the FA profile of HM between different regions and lactation stages, with differences in polyunsaturated FA levels being the most pronounced. Nearly 70% of sn-2 FAs were saturated FAs, of which C16:0 accounted for approximately 75%. C8:0, C10:0, C18:0, C20:0, C22:0, and all of the unsaturated FAs were mainly located at the sn-1 and sn-3 positions, while C14:0, C15:0, and C16:0 were mainly at the sn-2 position. The proportion of C12:0 and C17:0 at sn-2 was approximately equivalent to that at the sn-1, 3 positions. CONCLUSIONS: The results indicate the variability in the FA profile of HM between regions and lactation stages. The contents of polyunsaturated FAs and sn-2 FAs, especially palmitic acid, should be paid more attention when optimizing infant formula.

Identification and verification of differentially expressed genes in yak mammary tissue during the lactation cycle.

Yaks (Bos grunniens) live primarily in the Qinghai-Tibetan plateau (altitude: 2000-5000 m). Their milk presents unusual characteristics, containing large amounts of solids including fat and protein, and it is, therefore, important to understand the genetic makeup of the yak. To identify potentially critical genes playing a role in yak mammary tissue from colostrum to mature milk phase of lactogenesis, the early lactation (colostrum) stage (ELS; day 1 after parturition) and mature lactation (milk) stage (MLS; day 15) were chosen for comparison. An ELS-specific cDNA library was established by suppression subtractive hybridization and 25 expressed sequence tags at ELS were identified by sequencing and alignment. To further confirm our results the expression levels of 21 genes during the lactation cycle were measured using quantitative real-time RT-PCR (qRT-PCR). The qRT-PCR results confirmed 9 significantly up-regulated genes at ELS vs. MLS in yak mammary tissue, in which the l-amino acid oxidase 1 (LAO1) and collagen, type I, alpha I (COL1A1) were the most significantly up-regulated. During the lactation cycle, the highest expression of some milk fat genes (i.e., XDH and FABP3) in yak mammary tissue appears earlier than that in dairy cow. Our data also indicate MYC potentially playing a central role through putative regulation of COL1A1, CD44, SPARC, FASN and GPAM.

Parenteral Cu Supplementation of Late-Gestating and Lactating Iberian Red Deer Hinds Fed a Balanced Diet Reduces Somatic Cell Count and Modifies Mineral Profile of Milk.

: This study describes the effects that Cu supplementation of late-gestating and lactating females (hinds) of Iberian red deer fed a balanced diet have on milk production, composition, and somatic cell count (SCC). Experimental hinds (n = 9) were subcutaneously injected every 42 days with Cu (0.83 mg Cu/kg body weight) from day 202 of gestation until the end of lactation (week 18). Control hinds (n = 8) were injected with a physiological saline solution with the same volume and at the same frequency as the experimental group. Copper supplementation decreased the SCC from 1.64 to 1.36 log 10/mL (p = 0.003) and modified the milk mineral profile. In particular, milk from hinds supplemented with Cu had more Ca (p = 0.02), Mg (p = 0.06), and K (p = 0.03) than milk from control hinds. However, Cu supplementation did not influence the Cu content of milk. Therefore, it can be concluded that Cu supplementation of hinds fed a balanced diet from late-gestation to the end of lactation could be a good strategy to reduce SCC and modify the mineral profile of milk increasing its Ca content.

Lipidomics analysis for identifying the geographical origin and lactation stage of goat milk.

Goat milk samples of three lactation stages (colostrum, mature and late milk) were collected from three farms and analyzed with an untargeted method based on UPLC-Q-Exactive Orbitrap Mass Spectrometry and multivariate statistics. A total of 14 lipid subclasses and 756 lipid molecules were identified in samples. Five lipid subclasses and 51 lipid molecules in milk were significantly different among different geographical origins. Two lipid subclasses and 26 lipid molecules were significantly different among different lactation stages. Combined with the partial least squares discriminant analysis results of lipid molecules with a VIP value (Variable Importance in the projection) higher than 1, totally 38 and 19 lipid molecules could be used as potential indicators to identify geographical origins and lactation stages, respectively. Based on six and five selected molecules, the correct rates of discrimination models for geographical origin and lactation stage respectively reached 100% and 96%.

Energy-Protein Supplementation and Lactation Affect Fatty Acid Profile of Liver and Adipose Tissue of Dairy Cows.

This article addresses the hypothesis that lactation stage, parity and energy-protein feed additive affect fatty acid composition of blood, liver and adipose tissue of cows. The experiment was conducted on 24 Polish Holstein-Friesian cows divided into two feeding groups. One group of cows was fed solely a total mixed ration, while the other group was fed a ration with the addition of 2 kg of energy-protein supplement per cow/day. During the experiment, the samples of liver, adipose tissue and blood were taken and their fatty acid compositions were determined. Analysis of variance was applied to fatty acid relative weight percentage to determine the effect of the stage of lactation, parity, and energy-protein supplement on the fatty acid composition of the tissues. Stage of lactation had a significant impact on the content of many fatty acids in all examined tissues. We found that parity had no effect on fatty acid composition of blood, whereas it significantly affected C16:1 c9 in liver, and C16:1 c9 and C18:0 in adipose tissue. Energy-protein supplement significantly affected the content of most fatty acids in blood (e.g., C18:1 t11 and C18:3 n-3) and liver (C18:3 n-3, both isomers of conjugated linolenic acid and n-3 fatty acids derived from fish oil), but it did not affect the profile of the adipose tissue of cows. According to our best knowledge, this is the first study showing the relationship between parity, stage of lactation and the composition of fatty acids in blood, liver and adipose tissue of cows.

Transport of fatty acids within plasma lipoproteins in lactating and non-lactating cows fed on fish oil and hydrogenated palm oil.

The aim of this study was to elucidate the effect of dietary fish oil (FO) and a blend of FO and hydrogenated palm oil (FOPO) on transport of fatty acids (FA) within plasma lipoproteins in lactating and non-lactating cows. Two trials were conducted (one with lactating and another with non-lactating dairy cows) in two 3 × 3 Latin squares that included three periods of 21 days. Dietary treatments for lactating cows consisted of a basal diet (Control; no fat supplement), and fat-supplemented diets containing FO (500 g/day/cow) and FOPO (250 FO + 250 g/day/cow hydrogenated palm oil). For non-lactating cows, dietary treatments consisted of a basal diet (Control; no fat supplement), and fat-supplemented diets containing FO (170 g/day/cow) and FOPO (85 FO + 85 hydrogenated palm oil g/day/cow). In lactating cows, compared with control and FOPO, FO increased C16:0, C18:3 cis-9, 12, 15, C18:2 cis-9, trans-11 and total saturated and polyunsaturated FA in plasma and increased C16:0, C18:2 cis-9, trans-11, total polyunsaturated and total polyunsaturated n-6 in high-density lipoprotein (HDL), whereas in non-lactating cows, compared with control and FOPO, FO increased C16:0, C18:1 trans-11, C18:2 trans-9, 12, C18:2 cis-9, trans-11, C20:5 n-3 and total saturated and polyunsaturated FA in plasma; C16:0, C18:1 trans-11, C18:1 cis-9, C18:2 trans-9, 12, C20:5 n-3 and total monounsaturated FA in HDL; and C18:1 trans-6-8, C18:1 trans-9, C18:1 trans-10, C18:1 trans-11, C18:3 cis-9, 12, 15 and C20:5 n-3 in low-density lipoprotein (LDL). FO increased C20:5 n-3 in plasma and lipoproteins in non-lactating cows and increased C18:3 cis-9, 12, 15 in plasma (in lactating cows) and LDL (in non-lactating cows). We concluded from results of this study that in bovine plasma, the LDL fraction appears to be the main lipoprotein transporting C18:1 trans isomers and is more responsive than other lipoprotein fractions to variation in supply of dietary lipids.

Selenium and Antioxidant Status in Dairy Cows at Different Stages of Lactation.

Thirty-five multiparous Holstein cows averaging 550 ± 50 kg of body weight and in 2 to 4 parity were divided into three groups according to lactation stage (group A: nine cows from 4 to 1 weeks prepartum; group B: 11 cows from 1 to 30 days postpartum; group C: 15 cows from 30 to 100 days postpartum). Selenium concentration, malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) activity, thioredoxin reductase (TrxR) activity, and total antioxidant status (TAS) in serum were determined to evaluate selenium and antioxidant status in dairy cows at different stages of lactation. The results showed that mean serum selenium concentration, MDA level, and GSH-Px activity of cows in early lactation increased significantly (P < 0.05) when compared with cows in the dry period and peak lactation. Conversely, serum TrxR activity and TAS declined during this period (P < 0.05). The increase of serum MDA level during early lactation indicate that the reactive oxygen species, including lipid hydroperoxides, increase in this period, thus placing the cows at a greater risk of oxidative stress. The significant decrease in TrxR activity that is accompanied with a decrease in TAS during early lactation suggests that dairy cows have low antioxidant defense in this period and TrxR may be an important antioxidant defense mechanism in transition dairy cows.